EP1026504B1 - Verfahren zur Bestimmung von Rezeptorbindungseigenschaften und Reagenz zum Assay - Google Patents

Verfahren zur Bestimmung von Rezeptorbindungseigenschaften und Reagenz zum Assay Download PDF

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Publication number
EP1026504B1
EP1026504B1 EP00101326A EP00101326A EP1026504B1 EP 1026504 B1 EP1026504 B1 EP 1026504B1 EP 00101326 A EP00101326 A EP 00101326A EP 00101326 A EP00101326 A EP 00101326A EP 1026504 B1 EP1026504 B1 EP 1026504B1
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EP
European Patent Office
Prior art keywords
ligand
receptor
reagent
assay
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
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EP00101326A
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English (en)
French (fr)
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EP1026504A1 (de
Inventor
Yoshihiro C/O Tsuruga Institute of Biotechn Soya
Shigeaki C/O Tsuruga Institute of Biotech Nishii
Kazuhiro C/O Tsuruga Institute of Biotech Matsui
Takuya C/O Tsuruga Institute of Biotec Ishibashi
Yoshihisa C/O Tsuruga Institute of Bio. Kawamura
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Toyobo Co Ltd
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Toyobo Co Ltd
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/723—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
    • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
    • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
    • Y10S530/815—Carrier is a synthetic polymer
    • Y10S530/816—Attached to the carrier via a bridging agent
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/827—Proteins from mammals or birds

Definitions

  • the present invention relates to a method for assaying a receptor binding substance in an aqueous sample (e.g., biological material, sea water, river water, ground water and the like), or a method for assaying the receptor binding property of a chemical substance, and an assay reagent to be used for this method.
  • an aqueous sample e.g., biological material, sea water, river water, ground water and the like
  • an assay reagent to be used for this method.
  • the receptors present on the cell membrane are hormone receptors such as adrenergic receptor, luteinizing hormone receptor and the like. Many of these receptors are responsible for intracellular regulation through signal transmission and amplification via tyrosine kinase, adenylate cyclase and the like that are regulated by the interactions of the receptors and their ligands. Those present in a cell or nucleus are exemplified by estrogen receptor, retinoide receptor and the like.
  • Some receptors act as a carrier in blood, such as transferrin.
  • the action mechanism of nuclear receptor is considered to involve binding of a nuclear receptor and its ligand, which activates a nucleic acid binding region of the receptor, and binding thereof with the nucleic acid to control transcription and translation from the nucleic acid, which ultimately results in the control of various reactions in the body.
  • the binding of substance and receptor has been investigated and studied as models representing various reactions in the body.
  • the receptor is meant a substance which is not an antibody and which shows hormone binding property, biochemical messenger, steroid, drug, drug metabolite, polypeptide, protein, vitamin, alkaloid, monosaccharide, disaccharide, polysaccharide and the like.
  • the reported method using fluorescence depolarization by Bolger et al comprises competitively reacting a fluorescent-labeled tracer with a receptor and an assay target substance with a receptor, and measuring depolarization of the fluorescence due to the binding of the tracer and the receptor.
  • This method is defective in that the use of the fluorescent-labeled tracer causes lower reactivity and the method is subject to an influence of a contaminating fluorescent substance in the sample and the cloudiness of the sample.
  • the method using a radioisotope by Obourn et al comprises competitively reacting an RI-labeled tracer with a receptor and an assay target substance with a receptor, and quantitatively assaying the radioisotope bound with the receptor.
  • This method can be used only in a specific facility because it uses a radioisotope that limits facility and workability.
  • the method using surface plasmon resonance by Ward et al uses an immobilized receptor, and analyzes the direct molecular interaction between the assay target substance and the receptor.
  • This method fails to simultaneously process plural samples, and requires an expensive sensor chip and a special apparatus.
  • thermodynamic assay by Moore et al is superior in that it does not require immobilization of receptor or use of a labeled tracer. However, this method also fails to simultaneously process plural samples and requires a special apparatus.
  • the present invention is based on the finding that a ligand not bound with a receptor can be assayed alone with ease by the steps comprising competitively reacting a ligand and an assay target substance with the receptor in a solution, and, without physically removing the ligand bound with the receptor, further adding an antibody against the ligand and a labeled ligand to allow reaction.
  • the present invention provides the following.
  • the present invention in brief relates to a method for evaluating the receptor binding property of a natural compound or artificially synthesized compound. According to this method, the substance to be evaluated and an unlabeled ligand are competitively reacted at a binding site on the receptor, and the concentration of the free ligand is directly assayed in the reaction mixture, without separating the ligand bound with the receptor from the free ligand.
  • the receptor includes hormone, drug, drug metabolite, polypeptide, protein, saccharides, biochemical messenger, vitamin, ligand binding domains thereof, and the like.
  • the receptor embraces receptors on biomembranes (e.g., luteinizing hormone receptor, follicle-stimulating hormone receptor, human choriogonadotropin receptor, thyroid-stimulating hormone receptor and the like), nuclear and cytoplasmic receptors (e.g., estrogen receptor, androgen receptor, progesterone receptor, peroxisome amplification promoting receptor, glucocorticoid receptor, retinoic acid receptor, retinoid X receptor, thyroid receptor, ubiquinone receptor, vitamin D receptor, mineral corticoid receptor and the like) and receptors in serum as carrier (e.g., thyroglobulin, transferrin and the like).
  • biomembranes e.g., luteinizing hormone receptor, follicle-stimulating hormone receptor, human choriogonadotropin receptor, thyroid-stimulating hormone receptor and the like
  • nuclear and cytoplasmic receptors e.g., estrogen receptor, androgen receptor, progesterone
  • the ligand in the present invention means luteinizing hormone, follicle-stimulating hormone, human choriogonadotropin, thyroid-stimulating hormone, estrogen, androgen, progesterone, peroxisome, glucocorticoid, retinoic acid, retinoid X, thyroid, ubiquinone, vitamin D, mineral corticoid, iron and the like, which correspond to the above-mentioned receptors.
  • the binding ratio with the receptor of assay target substance and ligand is determined according to their existing ratios and respective binding constants.
  • the binding ratio of the ligand with the receptor becomes constant.
  • the amount of the ligand that binds with the receptor decreases depending on the amount of the substance and its binding constant, and the amount of free ligand increases.
  • the present invention most characteristically comprises the following steps to evaluate the receptor binding property:
  • the assay of an endocrine disrupting chemical has recently been highly demanded.
  • the combination of a receptor and a ligand for screening of such substance is preferably an estrogen receptor and estradiol, estrone or estriol, a thyroid hormone receptor and thyroxine or triiodothyronine and the like.
  • the assay method of the present invention can be used for the combination of a receptor and a ligand other than those mentioned above.
  • the above-mentioned ligand preferably has not undergone labeling, binding, processing or denaturing.
  • the amount of the receptor to be added is preferably such as allows for binding of not less than 50% of the coexisting ligand. When the concentration is less than 50%, the ligand shows very small variation in the concentration, which in turn makes the evaluation of receptor binding property difficult.
  • the concentration of free ligand is measured by a known method.
  • a spectroscopical method or a fluorescence/luminescence assay is applied.
  • the assay generally includes competitive reaction of the free ligand and an enzyme-labeled ligand with an immobilized anti-ligand antibody.
  • the enzyme to be used as a label may be peroxidase, alkaline phosphatase, ⁇ -galactosidase, acetylcholinesterase and the like, with preference given to peroxidase.
  • a fluorescent marker, a luminescent marker and the like are used instead of an enzyme label.
  • fluorescent marker examples include fluorescein, rhodamine and the like and examples of the luminescent marker include acridinium ester, photoprotein (e.g., aequorin) and enzyme (e.g., luciferase).
  • acridinium ester examples include acridinium ester, photoprotein (e.g., aequorin) and enzyme (e.g., luciferase).
  • the antibody to be used in the present invention is free of any particular limitation as to the origin, property and the like, as long as it can assay a ligand.
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • an antibody against the substance is preferably added to the reagent to obliterate an influence of the substance on the assay.
  • an antibody (secondary antibody) against the antibody to be used for higher orientation may be previously bound with a water insoluble carrier.
  • a known method for providing a solid phase such as a method comprising previously immobilizing protein A or protein G and binding the antibody, or binding a biotinylated antibody on a water insoluble carrier on which streptavidin or avidin has been immobilized in advance.
  • an organic solvent is used to dissolve the sample.
  • the organic solvent to be used is not particularly limited, preferred are methanol, dimethyl sulfoxide (DMSO), acetonitrile, ethanol and the like.
  • the antibody to be used needs to be resistant to the organic solvent to be used. Desirably, this antibody retains the antibody activity by 60% or more in an organic solvent having a 1% concentration.
  • the antibody activity here is meant the specific affinity thereof for the ligand to be used.
  • estrogen receptor 100 nM (in 10 mM phosphate buffer, 150 mM NaCl; pH 7.2) 17 ⁇ estradiol: 33 nM (in 10 mM phosphate buffer, 150 mM NaCl; pH 7.2)
  • Diethylstilbesterol (0, 3.3, 10, 33, 100, 333, 1000 ng/ml)
  • a sample (30 ⁇ l) and an estrogen receptor solution (20 ⁇ l) were mixed and 17 ⁇ estradiol solution (30 ⁇ l) was added, which was followed by incubation at 37°C for 1 hr.
  • the solution (50 ⁇ l) was dispensed to an anti-estradiol antibody-bound microtiter plate, simultaneously with which a horseradish peroxidase (HRP)-labeled estradiol solution (50 ⁇ l) was dispensed, which was followed by incubation at 37°C for 1 hr.
  • the reaction mixture was removed and the plate was washed 3 times with 10 mM phosphate buffer containing 0.15 M NaCl and 0.05% Tween 20.
  • Estrogen receptor 100 nM (in 10 mM phosphate buffer, 150 mM NaCl; pH 7.2) 17 ⁇ estradiol: 33 nM (in 10 mM phosphate buffer, 150 mM NaCl; pH 7.2) anti-estradiol antibody-bound microtiter plate A HRP-labeled estradiol
  • Estrogen receptor 100 nM (in 10 mM phosphate buffer, 150 mM NaCl; pH 7.2) 17 ⁇ estradiol: 33 nM (in 10 mM phosphate buffer, 150 mM NaCl; pH 7.2) anti-estradiol antibody-bound microtiter plate B HRP-labeled estradiol
  • diethylstilbesterol (0,1000 ng/ml; prepared with PBS, prepared with 1% DMSO solution)

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
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  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Endocrinology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Claims (12)

  1. Verfahren zur Bestimmung der Rezeptorbindungseigenschaft einer Assay-Zielsubstanz, das die folgenden Schritte umfasst:
    (a) kompetitive Umsetzung einer bekannten Konzentration eines Liganden und der Assay-Zielsubstanz mit einer bekannten Konzentration des Rezeptors in einer Lösung;
    (b) Messen der Menge des freien Liganden in der Lösung unter Verwendung von einem oder mehreren Antikörpern gegen den Liganden, ohne den an den Rezeptor gebundenen Liganden vor dem Assay physikalisch zu entfernen; und
    (c) Bestimmen der Rezeptorbindungseigenschaft der Assay-Zielsubstanz anhand der Menge des freien Liganden.
  2. Verfahren gemäß Anspruch 1, wobei der Schritt (b) die Zugabe von einem oder mehreren Antikörpern gegen den Liganden und eines markierten Liganden zu dem in Schritt (a) erhaltenen Reaktionsgemisch, so dass eine kompetitive Reaktion des Antikörpers und des freien oder markierten Liganden stattfinden kann, und das Messen der Menge des an den Antikörper gebundenen oder nicht gebundenen Markers umfasst.
  3. Verfahren gemäß Anspruch 1, wobei der Antikörper in einem 1%igen organischen Lösungsmittel seine Aktivität zu 60% oder mehr beibehält.
  4. Verfahren gemäß Anspruch 1, wobei der Ligand nicht markiert, gebunden, prozessiert oder denaturiert ist.
  5. Verfahren gemäß Anspruch 1, wobei der Rezeptor in einer Menge verwendet wird, die zu einer Bindung von 50% oder mehr des Liganden an den Rezeptor führt.
  6. Verfahren gemäß Anspruch 1, wobei der Rezeptor aus der Gruppe ausgewählt ist, die aus Rezeptoren von Hormonen, Wirkstoffen, Wirkstoffmetaboliten, Polypeptiden, Proteinen, Sacchariden, biochemischen Botenstoffen und vitamin- und ligandbindenden Domänen davon besteht.
  7. Reagens zur Bestimmung der Rezeptorbindungseigenschaft einer Substanz, umfassend ein Reagens, das eine bekannte Konzentration eines Rezeptors enthält, ein Reagens, das eine bekannte Konzentration eines Liganden des Rezeptors enthält und ein Reagens zur Messung eines freien Liganden, das einen oder mehrere Antikörper gegen den Liganden enthält.
  8. Reagens gemäß Anspruch 7, das weiterhin ein Reagens umfasst, das den markierten Liganden enthält.
  9. Reagens gemäß Anspruch 7, wobei der Antikörper in einem 1%igen organischen Lösungsmittel seine Aktivität zu 60% oder mehr beibehält.
  10. Reagens gemäß Anspruch 7, wobei der Ligand nicht markiert, gebunden, prozessiert oder denaturiert ist.
  11. Reagens gemäß Anspruch 7, wobei der Rezeptor in einer Menge verwendet wird, die zu einer Bindung von 50% oder mehr des Liganden an den Rezeptor führt.
  12. Reagens gemäß Anspruch 7, wobei der Rezeptor aus der Gruppe ausgewählt ist, die aus Rezeptoren von Hormonen, Wirkstoffen, Wirkstoffmetaboliten, Polypeptiden, Proteinen, Sacchariden, biochemischen Botenstoffen und vitamin- und ligandbindenden Domänen davon besteht.
EP00101326A 1999-01-25 2000-01-22 Verfahren zur Bestimmung von Rezeptorbindungseigenschaften und Reagenz zum Assay Expired - Lifetime EP1026504B1 (de)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP1598099 1999-01-25
JP1598099 1999-01-25
JP17453699A JP3649319B2 (ja) 1999-01-25 1999-06-21 レセプター結合能の測定方法および測定用試薬
JP17453699 1999-06-21

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EP1026504A1 EP1026504A1 (de) 2000-08-09
EP1026504B1 true EP1026504B1 (de) 2005-05-11

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US (2) US6638725B2 (de)
EP (1) EP1026504B1 (de)
JP (1) JP3649319B2 (de)
DE (1) DE60019988T2 (de)

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DE10028186A1 (de) * 2000-06-09 2002-09-19 November Ag Molekulare Medizin Verfahren zur Bestimmung der Menge von an Ziel-Molekülen gebundenen Liganden
DE10125258A1 (de) * 2001-05-23 2003-01-09 November Ag Molekulare Medizin Verfahren zur Bestimmung des Bindeverhaltens von an Ziel-Molekülen spezifisch bindenden Liganden
AU2003288650A1 (en) * 2003-01-08 2004-08-10 Pharmacia & Upjohn Company Llc Method for determining molecular affinities for human serum albumin
US7749445B2 (en) * 2005-05-02 2010-07-06 Bioscale, Inc. Method and apparatus for analyzing bioprocess fluids
GB2443694B (en) * 2006-11-10 2011-09-14 Platform Diagnostics Ltd Analyte saturation assay, methods and kits and devices
JP6795951B2 (ja) * 2016-11-09 2020-12-02 旭化成株式会社 濃度推定方法、検出装置、処理装置、プログラムおよび検出システム

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US20010046683A1 (en) 2001-11-29
US20040072260A1 (en) 2004-04-15
US6638725B2 (en) 2003-10-28
JP3649319B2 (ja) 2005-05-18
DE60019988T2 (de) 2006-02-23
JP2000283984A (ja) 2000-10-13
EP1026504A1 (de) 2000-08-09
DE60019988D1 (de) 2005-06-16

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